The invention relates to a pharmaceutical composition for treating blood coagulation disorders, in particular for treating factor VIII inhibitor patients. The invention furthermore relates to a method for preparing such a composition as well as the use thereof.
Blood coagulation is triggered by a series of consecutive reactions of different proteins and enzymes, respectively. By a deficiency of blood coagulation factors, the formation of fibrin from fibrinogen and, thus, wound closure, is prohibited; the consequence are hemorrhages. Such is the case with hemophilia A. This is the most wide-spread bleeding disease and is caused by a deficiency of factor VIII. For a substitution treatment of hemophilia A, preparations are used which contain factor VIII. Treatment with these preparations in most instances leads to a rapid hemostasis.
There are, however, also patients who do not only suffer from a factor VIII deficiency, but who have also developed an inhibitor directed against factor VIII. A further collective of patients has factor VIII inhibitors without suffering from hemophilia A Depending on the amount of factor VIII inhibitors present, the effect of factor VIII supplied is inhibited by neutralization of the latter.
At present, preparations on the basis of a plasma fraction which contains a mixture of coagulation factors are offered for a treatment of factor VIII inhibitor patients. This plasma fraction may, e.g., comprise the factors of the prothrombin complex (factors II, VII, IX and X). A blood coagulation-promoting preparation having factor VIII inhibitor bypass activity (FEIBA(copyright) TIM 4, from BAXTER AG) is, e.g., obtained according to AT-B 0 368 883 by treating cryosupernatant. This preparation also comprises the coagulation factors II, VII, IX and X.
The action of a FEIBA preparation is manifold due to its complex composition. Mariani et al. (Thrombosis Res. 31, 475-488 (1983)) mentions factor VII in its activated form as an active principle. It has been found that after infusion of a FEIBA preparation there occurs an increased content of factor VIIa in the plasma of hemophiliacs.
Likewise, Teitel (Thrombosis and Haemostasis 66 (5) 559-564 (1991)) discusses the role of factor VIIa in prothrombin complex concentrates with a factor VIII bypassing activity. At the same time also the active principle of factor Xa in such preparations is discussed. The prothrombin complex concentrates assayed contained factor VIIa, expressed by the ratio of factor VII activity to factor VII antigen, of 2.1 and 2.5.
The prothrombin-containing therapeutic composition prepared according to EP 0 044 343-B1 is suitable for the treatment of coagulation factor inhibitors and comprises an activated prothrombin complex in which the factors partially are activated The content of factor VIIa is from 8-80 units/ml The factor IX concentration ranges from 15 to 112 units/ml. Accordingly, the content of factor VIIa, based on factor IX, is 0.07-5.3 U of factor VIIa/U of factor IX. Vinazzer (Thromb. Res. 26:21-29 (1982)) shows the difference of the preparations AUTOPLEX, prepared according to EP 0 044 343, and FEIBA. As shown there, AUTOPLEX is characterized by the higher content of thrombin (factor IIa), measured in NIH units, as compared to FEIBA (cf. table 1, page 24).
Yet also highly purified factor VIIa preparations have been suggested for the therapy of coagulation inhibitor conditions (e.g. EP 0 082 182-B1) and Hedner et al. (Haemostasis 19, 335-343 (1989)).
An advantage of factor VIIa preparations is their freedom from factor VIII. The content of factor VIII in prothrombin complex preparations or in activated prothrombin complex preparations such as, e.g., FEIBA, has the effect in patients with functional factor VIII inhibitors that these inhibiting antibodies are boosted by a renewed administration of factor VIII with the consequence that the condition of the inhibitor hemophilia even deteriorates temporarily.
It has, however, been found that for an effective hemostasis in factor VIII inhibitor hemophilia, the action of factor VIIa preparations is interior to that of prothrombin complex factor preparations (Turecek P. et al., Thrombosis and Haemostasis, 1997, p.222).
It is thus an object of the present invention to provide a preparation which has the effectiveness or efficiency of prothrombin complex factor preparations, without, however, leading to the undesired immunological side reactions of such preparations.
The afore-mentioned object is achieved in that according to the present invention, an immunotolerant pharmaceutical prothrombin complex preparation comprising factors II, IX, X and, optionally, VII with a low factor VIII antigen content is provided.
The factor VIII antigen content of the preparation according to the invention preferably is less than 10%, in particular less than 5%. The factor VIII content preferably is less than 0.1 factor VIII:C antigen/U FEIBA. In a particularly preferred embodiment, the factor VIII antigen content is even below 0.03/U FEIBA, more preferred below 0.02/U FEIBA, and most preferred below the detection limit.
The finding that particularly in case of a further purification of the prothrombin complex factors from plasma or from a plasma fraction, the factor VIII content and, optionally, also the phospholipid content can be reduced so much while the activity of the prothrombin complex factors is largely retained must be considered to be surprising.
In particular, the pharmaceutical preparation according to the invention contains at least the factors IXa, Xa and VIIa and has FEIB activity, i.e. it shortens the coagulation time of a factor VIII-deficient plasma with a functional inhibitor (in this context, cf., e.g., AT-B 350 726).
The preparation according to the invention can be prepared from plasma or from a plasma fraction. The plasma fraction may be prepared from plasma, in particular plasma of human origin, by a chromatographic treatment, precipitation or centrifugation, or the supernatant of the cryoprecipitate is used.
The plasma fraction comprises vitamin K-dependent factors, such as factors of the prothrombin complex, yet also proteins S, C and/or Z are preferably contained therein.
In a particularly preferred embodiment, the preparation is free from phospholipids. Preferably, the upper limit of phospholipids contained is 0.1 nmol/U of FEIBA (for a determination, cf. the Examples).
Because of this freedom from phospholipids, the formation of undesired antibodies to factor VIII can be reduced or prevented, respectively.
According to the present invention, also a method or producing this preparation is provided. This method comprises the following steps:
a) Providing plasma or a plasma fraction comprising factors II, IX, X and, optionally, factor VII,
b) contacting the plasma or the plasma traction with a carrier material, optionally in the presence of a detergent, so that factor VIII and, optionally, phospholipids are separated from factors II, IX, X and, optionally, factor VII,
c) purifying the plasma or the plasma fraction, and
d) recovering a fraction comprising factors II, IX, X and, optionally, factor VII.
In a preferred variant, steps b and c or b, c and d, respectively, are carried out as one method step.
The plasma fraction used preferably is one having at least an intermediary purity.
By a xe2x80x9cpreparation having an intermediary purityxe2x80x9d, a plasma fraction is to be understood which is analogous to the definition of the intermediary purity of factor VIII preparations (in this context, cf., e.g., Wood Clive (ed.) Factor VIII: Purity and prophylaxis, Royal Society of Medicine).
Accompanying proteins which are not removed by a chromatographic purification thus may still be present in a preparation of intermediary purity.
As the Starting material, a conventional commercially available prothrombin complex factor preparation may be used, such as, e.g., FEIBA S-TIM 4, from BAXTER, or activated prothrombin complex.
As respective purification treatments, the methods known from the prior art are employed, preferably a chromatographic treatment, precipitation or centrifugation is carried out. As plasma fraction, also cryoprecipitate supernatant can be used.
The carrier material is a material suitable for chromatography, filtration and/or nanofiltration. The filtration in particular is an affinity or membrane filtration.
If a pre-purified material is used, e.g. a material pre-purified by means of an anion exchanger, a readsorption on a further carrier material, preferably on the same carrier material as has been used for the pre-purification, is used, under altered conditions.
In a preferred embodiment, the carrier material is a factor VIII-specific carrier material, in particular a matrix suitable for affinity chromatography. Particularly peferably a vWF-containing matrix is used.
To such a carrier material, factor VIII and, optionally, phospholipid are preferably adsorbed, while the factors II, IX, X and, optionally VII, are not bound.
The carrier material may, however, also be a carrier material non-specific for factor VIII, e.g. a weak anion exchanger, e.g. a DEAE, TMAE anion exchanger or further anion exchangers sufficiently known from the prior art.
Depending on the conditions chosen, either factor VIII and, optionally, phospholipids are adsorbed on the carrier material, while factors II, IX, X and, optionally, factor VII are riot bound, or vice-versa.
In a further preferred embodiment, the carrier material has a higher affinity for the prothrombin complex than for factor VIII. Thus, e.g., factors II, IX, X and, optionally, factor VII are adsorbed, while factor VIII and, optionally, phospholipids are eluted
Factor VIII may also be specifically inactivated and then may in such inactivated form, e.g., no longer bind to the carrier material. Such an inactivation of factor VIII may, e.g. be effected by dissociation by using e.g. a chelating agent, by degradation, in particular proteolytic degradation, e.g. by means of serine proteases, such as thrombin or activated protein C, by the binding of affinity partners, such as antibodies or peptides.
All the method variants disclosed may also be carried out in the presence of a detergent, in particular of a non-ionic detergent. Preferably, a polyether or a polysorbate is used as the detergent, in particular Tween or Triton is used.
If a detergent is present, in a preferred embodiment it is removed again or separated, respectively.
In a further preferred embodiment, a step of inactivation of possibly present pathogens, in particular selected from the group of heat treatment, vapor treatment, treatment with a solvent and/or treatment with a detergent, is provided.
The preparation according to the invention thus is generally obtainable according to a method described before. It is particularly suitable for producing a medicament which is suitable for the treatment of factor VIII inhibitor (=hemophilia A) patients, in particular for such patients who have an inhibitor titer of greater than 1 Bethesda U/ml plasma, preferably greater than 5 Bethesda U/ml plasma. It may also be produced by a combination of the highly purified individual factors II, IX and X as well as, optionally, factor VII.
Since a biological material, i.e. material derived from organisms or body liquids or microorganisms, may be contaminated with pathogens, such as, e.g., infectious molecules or microorganisms and viruses or pyrogens, respectively, various methods or inactivating or depleting, respectively, pathogens or pyrogens, respectively, have been developed.
Such methods include physical and/or chemical treatments, such as, e.g., diverse filtration methods (e.g. nano-, dia- or ultrafiltration), a heat treatment, treatment with an acid or a base, treatment with a detergent and/or an organic solvent, as well as treatment with OV light or with laser light. Also various combinations of such methods for the inactivation or depletion, respectively, of pathogens have often been suggested in the prior art.
From EP 0 197 554, e.g., a method for depyrogenizing and inactivating viruses in a biological or pharmaceutical product is known which comprises a treatment with a virus-inactivating and depyrogenizing agent, such as, e.g., an amphiphilic substance and/or a solvent, on a solid phase on which the product has been adsorbed. Following this treatment, the virus-inactivating and depyrogenizing agent is separated from the solid phase, the adsorbed product is washed and finally is eluted from the solid phase.
From EP 0 131 740, the treatment of a protein-containing composition in a solution with organic solvents, such as di- or tri-alkyl phosphates, optionally in the presence of a detergent (solvent/detergent treatment) is known, whereby protein compositions free from lipid-containing viruses can he obtained.
From AT patent 402,151, a heat treatment is known in which, prior to heating, a tenside at a concentration of at least 1% by weight is admixed to a preparation which is present in an aqueous solution.
A further method for reducing or suppressing, respectively, undesired activities in biological or pharmaceutical products is known from BP 0 083 999. This method is based on an extended contact with a solution or suspension of a non-denaturing amphiphilic agent. The depyrogenized product is treated with an ion exchanger to remove the amphiphilic agent.
A disadvantage of many of these methods known from the prior art is the frequent occurrence of losses of activity of the labile proteins, e.g. blood proteins, contained in the compositions to be treated. In particular when carrying out a chromatographic purification step, an inactivation of proteins occurs to a relatively high extent. A degradation of proteins may also lead to an activation. Thus, e.g., it is known that during a chromatographic purification, due to autokatalytic processes, factor VII is very easily activated to factor VIIa which is undesired because it is very labile.
A further disadvantage resides in great amount of time and apparatus required for many methods which greatly reduces their practicability and therefore frequently renders them unsuitable for application on a large scale.
Within the scope of the present invention, therefore, a method for effectively inactivating pathogens in biological materials, which is gentle on proteins, in particular on labile proteins, is to be used which can easily be adapted to a large scale and which can he carried out economically. In particular, a degradation and a possible activation of proteins susceptible therefor should largely be avoided in this method for inactivating pathogens.
In this method for inactivating pathogens, in particular viruses, in a biological material this material is incubated with a chemical agent, wherein the incubation is carried out in the presence of an eluotropic salt corresponding to a NaCl concentration of at least 200 mmol/l, preferably at least 300 mmol/l.
Inactivation of pathogens in solution offers some advantageous over the treatment of an adsorbent. Thus, e.g., the practicability of such a method is higher in a homogenous, single-phase system, and validation of the inactivation step is better possible. Moreover, the better accessibility of pathogens in a relatively homogenous phase seems to increase the efficiency of the method step.
The biological material preferably comprises a human protein and, in particular, it is plasma or a plasma fraction or it is derived from a cell culture. Preferably, the biological material comprises a blood factor, such as factors XII, XI, VIII, V, von Willebrand factor or fibrinogen, in particular a vitamin-K-dependent protein, such as factor II, factor VII, factor IX, factor X, protein C, protein S or protein Z, respectively.
The proteins may be present as single factors, preferably in purified form, or in a complex mixture. In a particularly preferred embodiment, the biological material comprises at least one factor of the prothrombin complex and is particularly a prothrombin-complex-containing traction or a factor VII-containing material, e.g. after a cryoprecipitation of plasma, one starts from the respective supernatant (cryosupernatant) thereof.
The preparation according to the invention preferably is one having FEIB activity (Factor Eight Inhibitor Bypassing Activity), i.e. a preparation which is suitable for treating factor VIII inhibitor patients.
The cell culture-derived material preferably is a material comprising recombinantly produced blood factors, among them factors of intrinsic or extrinsic coagulation, of fibrinolysis, of thrombolysis, or the inhibitors thereof, in particular vitamin K-dependent blood factors. As cells, the cells commonly employed for the expression of recombinant proteins can be used, preferably mammalian cells, such as, e.g., Vero, CHO or BHK cells. The respective proteins can be subjected to the inventive method for inactivation of possibly present pathogens either directly from the crude cell extract, or it may also be a pre-purified cell fraction.
The chemical agent is, e.g., a detergent (amphiphilic agent, tenside), which preferably is contained in an amount of at least 1%, more preferred more than 5%, most preferred more than 10%, yet, according to the invention also other chemical agents may be employed, in particular such which are already known to have a virucidal, bactericidal or depyrogenizing effect, or mixtures of the most varying chemical agents, respectively.
The choice is, however, limited in that the nativity of the biological material shall not be substantially adversely affected. For an economical mode or procedure, a chemical is chosen which retains more than 50% of the biological activity of the material, based on the activity prior to incubation, preferably at least 70%, in particular more than 85%. Retention of the biological activity means that the proteins contained in the biological material can fulfill their naturally ascribed function or the different functions, respectively. This biological activity may then be determined and stated depending on the type of protein, e.g. by means of a standardized chromogenic assay or by antigen determination. Optionally, the chemical agent is removed after incubation.
By detergent, quite general a synthetic, organic, surface-active substance is to be understood.
Preferably, a non-ionic detergent is used in the method according to the invention. Non-ionic tensides, such as polyether, in particular alkyl phenol polyglykol ether, are i.a. products of ethoxylation of fatty acids, fatty acid amides, fatty amines, fatty alcohols, aminoxides, fatty acid esters of polyalcohols and sugar esters.
Such a tenside does not have a denaturing action on the proteins and preferably is selected from the group of polysorbate and Triton. As the polysorbate, e.g., Tween(copyright) is used.
If detergents are used as the chemical agents, according to a preferred embodiment the former are used without addition of other agents, in particular without the addition of toxic organic substances or solvents, such as, e.g., TNBP In this manner, a risk or contamination is reduced to a minimum.
According to the method of the invention, the biological material is incubated with a chemical agent. Incubation means contacting the biological material with a solution, suspension or emulsion of a chemical agent for a period of time sufficiently long to inactivate possibly present pathogens or pyrogens, at a certain temperature. Contacting may, e.g., be effected by simply allowing the mixture to stand for a defined period of time.
Incubation is effected according to the present invention in the presence of an eluotropic salt. By xe2x80x9celuotropic saltxe2x80x9d in the following the salt in mixture with chemical agents or the salt in a complex composition is to be understood with the property of dissolving adsorbed substances out of solid or liquid-impregnated, also gel-type adsorbents and/or of displacing them. Preferably, the eluotropic salt is a desorption agent, as is employed in chromatographic methods. The adsorbed substance is i.a. sufficiently soluble in the presence of the eluotropic salt, i.e. preferably conditions are chosen which do not precipitate the biological material.
The type and concentration of the salt or of the composition, respectively, is generally chosen in dependence on the adsorbent used. The eluting action of the salt, e.g., depends on the polarity of the solvent, i.e. it increases e.g. in the sequence ethanolxe2x80x94acetonexe2x80x94methanolxe2x80x94water. The adsorbent can be a solid phase, in particular a matrix suitable for ion exchange chromatography. In the composition containing the eluotropic salt also further additives may be contained, e.g. further salts. Preferably, the composition is an aqueous composition hating a pH in the range between 6.0 to 8.0, preferably around 7.0.
In a preferred embodiment, sodium chloride is used as the eluotropic salt, yet also other alkaline or alkaline earth salts, among them CaCl2, are used. As the eluotropic salts, also so-called chaotropic agents, such as, e.g., urea, rhodanides or guanidinium can be employed. The concentration of the salt is at least xe2x89xa7200 mmol/l, preferably xe2x89xa7300 mmol/l. The upper limit for the concentration used will particularly depend on the solubility of the respective salt and for NaCl, e.g., it is around 2 mol/l. Chaotropic substances, such as, e.g., urea, may optionally be used even up to a concentration of 8 mol/l.
Incubation of the biological material with the chemical agent is effected for a period of time sufficiently long to inactivate any pathogens possibly present, preferably for a period of time of between 10 min and 10 h, most preferred between 1 h and 5 h. The time required for the method according to the invention can be determined by means of model viruses, such as HIV, Sindbis, TBE or hepatitis viruses in a pre-assay.
Also the choice of temperature influences the period of time to be used. In the method according to the invention, incubation is preferably effected at room temperature, e.g. in a temperature range of between 15 and 45xc2x0 C., in particular between 20 and 30xc2x0 C.
In the method according to the invention, the biological material preferably is adsorbed on a solid carrier, purified, and incubation is carried out immediately after elution of the purified material. Elution and incubation may be carried out consecutively, they may, however, also occur simultaneously.
According to a further preferred embodiment, incubation is carried out after a chromatographic purification of a biological material, the eluate having been further processed, e.g. by centrifugation, filtration or other physical methods.
The solid carrier preferably is a material suitable for chromatography, in particular a material suitable for ion exchange chromatography, hydrophobic chromatography or affinity chromatography. Materials such as Sepharose(copyright), Superdex(copyright), Sephadex(copyright), Spherodex(copyright), Toyopearl(copyright), or inorganic materials, such as hydroxyl apatite, are, e.g., used.
As the ion exchanger, anion exchange materials, such as, e.g., DEAE-Sephacel(copyright), DEAE-Sephadex(copyright), DEAE-Sepharose(copyright) CL6B DEAE-Sepharose(copyright)Fast Flow, QAE-Sephadex(copyright), Q-Sepharose(copyright)Fast Flow, Q-Sepharose(copyright) High Performance, DEAE-Tris-acryl, DEAE-Spherodex(copyright), Q-Hyper-D (obtainable through Sepracor), DEAE-Toyopearl(copyright), QAE-Toyopearl(copyright), Fractogel(copyright) EMD-TMAE or other Fractogel materials can be used.
As examples of hydrophobic chromatographic materials, e.g. butyl-Sepharose(copyright), octyl-Sepharose(copyright), phenyl-Sepharose(copyright), Fractogel(copyright)TSK-butyl, t-butyl-HIC Support or TSK Gel Butyl Toyopearl(copyright) should be mentioned.
The biological material may be adsorbed directly on the carrier from a complex mixture and purified, the inactivation step, may, however, also be preceded or followed by further steps for purifying the material, further chromatographic purification steps being preferred within the scope of the present invention.
By the method according to the invention, pathogens are inactivated. By pathogen, also fragments of, e.g., viruses, in particular also the isolated genome or its fragments, are understood.
The pathogens may be lipid-enveloped pathogens, such as, e.g., hepatitis B; virus, or non-lipid-enveloped pathogens, such as, e.g., hepatitis A virus.
At present, virus inactivation methods are called effective, if after using the method on a sample of a biological material which had been admixed with a high dose of a test virus, e.g. HI virus or Sindbis virus as a model virus for hepatitis viruses, viruses cannot be detected any longer in the sample, the virus titer thus having been reduced to below the detection limit. Detection and quantitation of nucleic acids may, e.g., be effected by means of a PCR method as described in AT patent 401,062, or by direct titration.
As a measure of inactivation, the so-called reduction factor is known which, after a single addition of test virus, is calculated from the decadic logarithm of the quotient of starting and final virus titers. From the European Guideline EC III/8115/89-EN of the Commission of the European Communities, furthermore the so-called total reduction factor is known. It is calculated from the sum of the reduction factors of individual, subsequent inactivation methods.
Also a further, independent step for inactivating or depleting, respectively, pathogens is preferably carried out. For this, all the methods known from the prior art can be used, to minimize the risk of infection.
In particular, a filtration and/or a heat treatment is effected as a further inactivation or depletion step.
As the filtration, preferably a nanofiltration is performed. A preferred heat treatment is carried out on solid biological material, e.g. on a lyophilisate having a controlled water content, e.g. a water content of between 5 to 8%, and at a temperature of between 50 and 80xc2x0 C., as described in EP-0 159 311.
In a preferred embodiment, a 2-step treatment with a detergent as the chemical agent is provided. Therein, a detergent in an amount of at least 1%, preferably at least 5%, most preferred at least 10%, is used in a first step. In a second step, a further detergent is used in an amount of at least 10%, preferably at least 12%, most preferred at least 14%. The detergent used may be the same one for both steps, however, also different detergents may be used. Quite generally, the risk of a virus infection after administration of a corresponding preparation can be highly reduced or excluded by the combination of steps for virus inactivation.
According to the present invention, also a chromatographically purified preparation is provided, comprising an autodynamically activatable blood factor having a portion of activated blood factor of less than 50%, based on the content of activated and non-activated blood factor, preferably less than 40%, more preferred less than 30%, even more preferred less than 20%, further preferred less than 10%, most preferred less than 1%, and a detergent content.
In particular, the preparation is a prothrombin complex-containing preparation comprising a factor VIIa activity of less than 50%, based on the content of activated and non-activated factor VII, preferably less than 10%, most preferred less than 1%. The detergent content of the preparation according to the invention is in a pharmaceutically acceptable amount, preferably between 1% and the detection limit of the detergent.
By xe2x80x9cautodynamically activatable blood factorxe2x80x9d according to the present invention a blood factor is to be understood which is autocatalytically activatable, by surface contact or by processes, such as, e.g., chromatographic processes. In particular, such a blood factor is a factor selected from the group of factor VII, factor XII, factor XI and pre-Kallikrein.
In a Further preferred embodiment, the preparation is free from serine protease inhibitors, such as, e.g., thrombin inhibitors, and their cofactors, such as, e.g., heparin. In a special embodiment, the freedom from such substances is already given during a chromatographic process.
Therefore, the present invention also relates to corresponding preparations, obtainable by the method according to the invention.
In the preparation according to the invention, also further additives may be contained, e.g. stabilizing substances, such as amino acids.